Animals: We employed Foxp2tm1.1(cre)Rpa/J transgenic mice in which IRES-Myc tag-nuclear localization signal (NLS)-cre-GFP-frt-neomycin-frt were introduced just after the termination codon of the mouse Foxp2 gene via homologous recombination. The mutation was created via homologous recombination in (129S6/SvEvTac x C57BL/6) F1-derived G4 embryonic stem (ES) cells. The frt-flanked neomycin cassette was excised through crosses with animals that broadly expressing Flp recombinase. The GFP is believed to be nonfunctional. Resultant mice were backcrossed to C57BL/6J for 9 generations by the donating laboratory to the Jackson laboratory (Strain #:030541; RRID:IMSR_JAX:030541). All transgenic mice used here were heterozygous for the transgene and backcrossed to the C57BL6 strain and wildtype littermates were used as controls. We bred these mice in our animal facility and confirmed their genotype by using a Red Extract N-amp Tissue PCR kit (Sigma Aldrich; Catalog # XNAT-1000RXN) and Cre forward and reverse primers to detect the Cre recombinase gene. Their wildtype litter mates were used as controls, in each experiment.
Validation of mice: To test if the Cre expression was eutopic with FoxP2 expression FoxP2-Cre mice from Jackson Labs, we also validated by crossing them to the L10 reporter mice. The cre-positive neurons were labeled with green fluorescent protein (GFP), and when the tissue was immuno-stained for FoxP2, we could observe nearly all green (GFP) neurons expressed labeling for Fox-P2 (red) in their nuclei (supplementary Fig.2a-d). This confirmed that we could reliably use these mice for expressing cre-dependent virus vectors in the FoxP2 neurons in the PB, and then use fiber-photometery or optogenetics to record their activity profiles or manipulate them (examples of cre dependent transfections- Fig.2b,4b and 6c). PBFoxp2 neurons did not overlap with the PBCGRP neurons in the lateral PB.
All mice used in these experiments were male because female mice of the same age are smaller, and including animals of various sizes would introduce noise into analysis of respiratory volumes (which scale with body size) across groups. Animals were maintained on a 12 h light/dark cycle with ad libitum access to water and food and were singly housed after surgery, with ambient temperature of 21-23o C and humidity levels between 40-60%. Male littermates were randomly assigned to the experimental groups. All animal procedures met National Institutes of Health standards, as described in the Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee.
Vectors: For fiber-photometry we used AAV-pSyn-GCaMP6s from Addgene_100843. For optogenetic experiments, we used the excitatory opsin (AAV-EF1a-DIO-hChR2(H134R)-mCherry- AAV-serotype8) and the optogenetic neural silencer AAV-CAG-FLEX-ArchT-GFP (AAV-serotype-8). These viral vectors was procured from the University of North Carolina (UNC) vector core and has been previously used for excitation as well as for silencing neurons and their terminals by us 19,55. The viral vectors expressing excitatory and the inhibitory opsins were packaged at the UNC vector core.
Surgery: Under surgical anesthesia, mice were instrumented for sleep with implantation of EEG and EMG electrodes in addition to implanting either the fiber photometry cannula (unilateral), or integrated grin lens with baseplate (unilateral) or bilateral optical fibers targeted to the PB area (AP: -5.1 to -5.3mm; DV- 2.6mm; ML: ±1.3mm). All implants were done after 5 weeks post injection of the injections of the viral vectors in the PB, to ensure optimal expression of the gene expression. After recovery from surgery, mice were recorded for sleep and respiration after acclimatizing them to the recording apparatus for at least once in a week before the actual recordings were performed. We recorded the arousal and respiratory responses to CO2 by placing them in the plethysmograph and recording them for both sleep and breathing by the procedure described previously19,21,55.
Experiment 1a: C57BL/6J mice (n=8) were moved from the animal housing room and placed in the plethysmographs where they were exposed continuously to either hypercapnic (21% O2; 10% CO2; 69% N2) or room air (21% O2; 0% CO2; 79% N2) for two hours, after first habituating them to the chamber at room air for two hours. Air or hypercapnic exposures were performed between 10:00 and 12:00. At the end, mice were deeply anesthetized with chloral hydrate (500mg/kg, ip) and transcardially perfused with saline, followed by 10% formalin. Brains were removed, post-fixed overnight immersed in 20% sucrose and cut into four alternate series of 30 micron frozen sections. After immune-staining the tissue for cFos and FoxP2, using specific antibodies as per the methods described below, the cells that were double labeled for cFos and Foxp2 were counted in the PBcl (0.5mmX 0.6mm placed dorsal to the single labeled cFos in the PBel, as in Fig. 1a2-d3) and KF (0.5mm X 0.5 mm placed ventral to the ventral spino-cerebellar tract, as in Fig. 1a1-d1) sub-nuclei, after scanning the slides using a slide scanner (Olympus VS200 slide scanner) and acquiring the images in Olympus OlyVia 3.3 software using square grids. To avoid counting bias, the cell counts were performed by investigators blind to the treatment groups (NL and JDL). The original figures in Fig1a-d were taken by a confocal laser microscope (LasX Leica DMi8 confocal). We counted the sections that were separated by at least 120µm, and this also involved counting only the nuclei for both the FoxP2 and the Cfos, that reduced the chances of counting cells twice. Cell counts were also corrected for the cell size using the Abercrombie correction factor.
Experiment 1b: Fiber-photometry implants: In vivo calcium monitoring during sleep-wake and with CO2 exposures was performed using fiber photometry. After 5 weeks of the viral injection of AAV-pSyn-GCaMP6s in the PB, laser light was passed into the brain via low-fluorescence fiber optic patch cord (0.48NA, Doric Lenses) connected to the implanted fiber optic cannula with a metal sleeve (Doric Lenses, MFC_400/430-0.48_5mm_MF-1.25). Using the patch cord, we simultaneously delivered light via LED drivers at 465 nm and 405 nm (Doric Lenses, CA), to measure the calcium-dependent and calcium-independent (UV, isobestic), excitation of the GCaMP. Excitation emission from the GCaMP protein passed back through the fiber optic patch cord and through the fluorescence Mini Cube (Doric Lenses) and detected by a photo-receiver (Doric Lenses). Signals detected by the photo-receiver were transmitted to Axon Digidata 1322A analog-to-digital converter and the signals were acquired using Axoscope software- v10 (Molecular Devices, Foster City, CA, USA), alongside the EEM/EMG and breathing signals. We used exported files to Spike2 and analyzed the respiratory signals using the spike respiratory scripts (Resp80t, Spike2, CED, UK) that were then correlated with the GCaMP activity.
The GCaMP6 raw data was normalized to the baseline fluorescence of each trial to obtain the ΔF/F (change in fluorescence intensity relative to the baseline fluorescence intensity) for different animals. The GCaMP ΔF/F values per second was calculated for 15 s before and during CO2 for each trial and statistically compared, along with RR and VT values for similar periods. The peak values of GCaMP ΔF/F, and the latency to peak during the entire 15s with CO2 exposure were also calculated. Two-way ANOVA was performed to compare the effects between pre and post CO2 exposures on the GCaMP ΔF/F and also for RR and VT.
Experiment 1c: GRIN lens implants: Foxp2tm1.1(cre)Rpa/J mice (heterozygous FoxP2-Cre), after 3weeks of the virus injection (AAV-pSyn-GCaMP6s) in the PB were implanted with a microendoscopic ProView™ Integrated Lens 0.6mm x 7.3mm (Inscopix Catalogue #1050-004413) that allowed for visualizing the activity during the lens implant. The lens was targeted to be ~200-300 µm above the neurons using the following coordinates- -5.0mm posterior to bregma, -1.4mm lateral from midline and -2.8 to 3.0 mm ventral to the dura mater. The baseplate provide the interface for attaching the miniature microscope during the calcium-imaging experiments, but during other times a baseplate cover (Inscopix catalogue # 100-000241) was attached to prevent damage to the micro endoscopic lens. Out of approximately 8 mice injected with GCaMP6s virus, 4 had successful implants and were used for the study.
Calcium imaging: We imaged the calcium activity at 5 frames per second, 200-ms exposure time at 20-30% LED power using the miniature microscope from Inscopix (nVista). These parameters caused minimal bleaching, and allowed long term recordings in mice. Mice were recorded for 5 min every hour for correlating the calcium activity to the 12h sleep-wake behavioral data. For correlating to the CO2 induced respiratory changes, mice were recorded in a plethysmograph, where they were subjected to repeated stimulus of 30s of 8% CO2 every 5 minutes, and the imaging was done for 4 trials (~20 min) every hour for 3h after 3-4h of habituation of mice to the recording chamber with every recording. This protocol caused minimum bleaching and allowed repetitive 2-3 recordings in an animal with a week of separation between subsequent recordings.
Experiment 2: Optogenetic activation of the PBFoxP2 neurons: For selective activation of the PBFoxP2 neurons, we injected an adeno-associated viral vector expressing the excitatory opsin AAV-FLEX-ChR2-mCherry in the PB (AP: -5.1 to -5.3mm; DV- 2.6mm; ML: ±1.3mm) and implanted these mice (n=10) with bilateral optical fibers targeting the lateral PB. We also injected some wild type mice (n=3) with AAV- FLEX-mCherry as well, which served as control, we did not observe any expression of ChR2. We recorded these mice for sleep and respiration when they were exposed to the room air as per the method previously described. Mice were subjected to 5 or 10s of 467nm laser stimulus with pulse width of 10ms and with frequencies of either 5Hz or 10Hz or 20Hz, in a random order and with each treatment separated by at least 7-10 days.
Experiment 3: Optogenetic inhibition of the PBFoxP2 neurons: A separate set of FoxP2 mice were injected in the PB with AAV-FLEX-ArchT-GFP (n=12), and to test whether PBFoxP2 neurons mediate CO2 induced changes in respiration, these mice were bilaterally implanted with optical fibers targeting the PB (AP: -5.1 to -5.3mm; DV- 2.6mm; ML: ±1.3mm) for the inhibition of the PBFoxP2 neurons. At 5 weeks post injection, these mice were recorded for sleep and breathing in plethysmography chamber, where the arousal and respiratory responses were assessed while they were subjected to repetitive CO2 stimulations as shown earlier 19,20,55,56.
Histology: At the conclusion of the experiments, the animals were perfused with 0.9% saline followed by 10% buffered formalin while under deep anesthesia. Brains were harvested for analysis of the effective location of the injection site. Brains were kept in 20% sucrose for 2 days and sections were cut at 30mm using a freezing microtome in four 1:4 series.
Immunohistochemistry: C-Fos: Oncogene Sciences, cat # Ab5, rabbit polyclonal, raised against amino acids 4–17 of human c-Fos. This antibody stained a single band of 55 kDa on Western blots from rat brain (manufacturer’s technical information). Sections were processed for detection of c-Fos alone or c-Fos in combination with FoxP2. All incubations were performed on free-floating tissue sections at room temperature. Sections were first incubated overnight in c-Fos antibody. When c-Fos staining was to be combined with FoxP2, we used Rabbit anti-Fos antibody (diluted 1:10K in PBS with 0.2% Triton X-100). After rinsing, sections were incubated in Alexa488 (green fluorescence) conjugated donkey anti Rabbit-IgG (Invitrogen, A11055) at 1:500 in PBS containing 0.2% Triton-X and 2.5% normal donkey serum for three hours. After rinsing in PBS sections were next incubated overnight with rabbit anti-FoxP2 (diluted 1:5000 in PBS with 0.2% Triton X-100 and 2.5% normal donkey serum). After rinsing the next day sections were incubated in Cy3 (red fluorochrome) conjugated donkey anti-rabbit IgG (Jackson Immuno-Research Labs, code#111-165-003) or with streptavidin-pacific blue (1:200, ThermoFischer, cat-S11222) after reacting the tissue to the appropriate biotinylated secondary antibody.
Mice injected with either GCamP6s or ArchT or ChR2, were immunostained either for GFP (Rabbit anti-GFP, 1:10K, Molecular Probes Cat# A-11122, RRID:AB_221569) or with mcherry (rabbit anti DsRed. 1:2K, Clontech, Cat-632496 ) as per standard immunohistochemistry protocols described previously19,55. These were then double stained for FoxP2 using either anti-rabbit (Rabbit anti-Foxp2, 1:10K, Abcam Cat# ab16046, RRID:AB_2107107) or anti-sheep antibodies (Sheep anti FoxP2, 1:5K, R and D Systems Cat# AF5647, RRID:AB_2107133). We used rabbit polyclonal antibody raised against a Synthetic peptide conjugated to KLH derived from within residues 700 to the C-terminus of Human FOXP2 (AbCam) and for the sheep polyclonal antiserum raised against recombinant human FoxP2 isoform 1, Ala640-Glu715, Accession # O15409 (R&D Systems) and these have been previously used and validated by others 57–59. Neither of these antibodies showed immunostaining when the primary antibodies were omitted, and when the tissue from control mice was used that were not injected with viral vector. Some of the brains (n=3) from WT mice were injected with CTb and were immunostained using Goat anti CTb (1:30K, Cat# 703, RRID: AB_10013220, List Biological Laboratories Inc., CA). Sections for double staining for GFP, mcherry, CTb or FoxP2 were incubated in fluorescent-labeled secondary antibodies (Alexa- 488 at 1:200 or Alexa- Cy3 at 1:200; Catalog #- A32790 and A10521, RRID- AB_2762833 and RRID- AB_2534030, Molecular probes, Thermo-Fischer Scientific) or with streptavidin-pacific blue (1:200, ThermoFischer, cat-S11222) for 2h and cover-slipped with fluorescence mounting medium (Dako, North America). When acquiring confocal images, sometimes pseudocolors were used to enhance clarity.
Fluorescent In situ hybridization (FISH using RNA Scope): We identified FoxP2 neurons by using Foxp2tm1.1(cre)Rpa/J mice crossed with R26-lox-STOPlox-L10-GFP reporter mice (FoxP2-L10, n=3), and labeled for Calca (the gene for CGRP) by using a set of FISH probes with RNAScope in brain sections from the KF and PBcl areas. The brain was sectioned at 30 µm and sections were mounted on glass slides in RNAs-free conditions, and RNA scope was performed using the multiplex fluorescent reagent Kit V2 (Cat# 323100, Advanced Cell Diagnostics, Hayward, CA). Brain sections on the slides were pretreated with hydrogen peroxide for 20 min at room temperature and then with target retrieval reagent for 5 minutes (at temperature above 99°C), followed by dehydration in 90% alcohol and then air-dried for 5 minutes. This is followed by a treatment with protease reagent (Protease III) for 30 minutes at 40°C. After rinsing in sterile water, sections were hybridized in CGRP/ Calca FISH probe (smFISH probe: Mm- Mm-Calca-tv2tv3-C1- Mus musculus, Calcitonin-related polypeptide alpha, transcript variant 2 mRNA; probe region: 63 – 995 Accession No. NM_001033954.3; Catalog# 420361; Advanced Cell Diagnostics, Hayward, CA) for 2 hours at 40°C, and this probe has been used previously to selectively label CGRP on the brain tissue 60,61. Sections were then incubated in 3 amplification reagents (AMP) at 40°C (AMP1 for 30minutes, AMP2 for 30 minutes and AMP3 for 15 minutes) followed by Horse radish peroxidase –C1amplification at 40°C for 15 minutes. Sections were then incubated in tyramide signal amplification (TSA) reagents with Cy3 fluorophore (Cat# NEL744001KT, Perkin Elmer, 1:1000) for 30 min to amplify and visualize CGRP mRNA in red. In the final step, sections were subjected to HRP blocking for 15 min at 40°C. After each step, sections were washed with 1X wash buffer provided in the kit. Following the CGRP RNAscope in-situ hybridization, immuno-labeling of GFP was performed on the same sections, as in-situ procedure quench the green fluorescence. For this, the brain sections were incubated in rabbit anti-GFP (1:1500), (Cat#A6455; Lot#1220284; Molecular probes) for overnight at 4°C, washed in PBS (3X2 minutes) and then incubated in secondary antibody (Alexa Fluor- 488 Donkey anti Rabbit, Life Technologies, Cat# A-21206) for 2h at room temperature. Finally, the slides were dried and cover-slipped with Dako fluorescence mounting medium (Cat# S302380-2, Agilent, CA), and scanned for analysis.
Data acquisition
All recordings were done at five weeks after injection of the viral vectors. All sleep and respiration recordings were done in a plethysmography chamber (unrestrained whole-body plethysmograph, Buxco Research Systems) which allowed us to record the breathing of the mouse while continuously monitoring the gas in the chamber. Electroencephalogram (EEG) and electromyogram (EMG) were recorded using Pinnacle preamp cables connected to an analog adaptor (8242, Pinnacle Technology). Gas levels in the chamber were continuously monitored using CO2 and O2 monitors from CWE, Inc (Ardmore, PA, USA). EEG, EMG, respiration, and CO2 and O2 levels were fed into an Axon Digidata 1322A analog-to-digital converter and the signals were acquired using Axoscope software- v10 (Molecular Devices, Foster City, CA, USA), or by acquired by the 1401 (CED, Cambridge, UK) and Spike2 ver.7 (CED, Cambridge, UK). Mice were connected to cables for sleep recording as well as with the fiber-optic cables connected to the pre-implanted glass fiber in mice, for transmitting the laser light.
Mice were also placed in the plethysmography chamber beginning at 9:00 A.M. for 6 h during their lights-ON and behaviorally inactive period, on each test day for these recordings. Here, they underwent either the Laser-ON or Laser-OFF protocols, separated by a week and in random order.
During the Laser-ON protocol, either 473nm or 593nm laser was ON for 5, 10 0r 60s followed by 5 mins off. With 473nm, the photo-stimulations were done at either 5, 10 or 20Hz with pulse width of 10ms, and these stimulations were done for either 5 or 10s on different days, with a period of 6-7days between each treatment. During photo-activation using the 473nm laser, only normocapnia air was used in the chamber. The inhibitory 593nm laser stimulations were continuous for 60s, and preceded each 30s of CO2 stimulus by 20s and lasted 10s after the hypercapnia stimulus. In the Laser-OFF condition, everything was the same, except that the laser light was not turned on. The gas input for the plethysmograph was switched either to normocapnic air (21% O2, 79% N2) or hypercapnic air (10% CO2, 21% O2, and 69% N2) for 30 sec with 5 minutes in between the two hypercapnic stimuli. For both the photo-activation and inhibition experiments, trials were analyzed for latency to arousal and respiratory changes only for those epochs where the mouse was in NREM sleep for at least 30 s before the stimulus onset.
Laser light: Mice were allowed at least 2d to acclimate to fiberoptic cables (1.5 m long, 200 µm diameter; Doric Lenses, Quebec, QC, Canada) and connecting interfaces coated with opaque heat-shrink tubing before the experimental sessions. During Laser-ON experiments, light pulses were programmed using a waveform generator (Agilent Technologies, catalog #33220A, CA, USA) to drive either 10ms of 473nm (blue laser, Laser Glow, Toronto, ON, Canada) pulses at 5, 10 or 20Hz, or drive the orange-yellow light laser (593 nm; Laser Glow, Toronto, ON, Canada) to be continuously on for 60s beginning 20 s before the onset of the CO2 stimulus. We also used the splitter - TM105FS1B (Thorlabs, NJ) to split the laser stimulus for bilateral activation or inhibition. We adjusted the laser such that the light power exiting the bilateral fiber-optic cables was 8-10 mW, and this was checked before and after the experiment. The light power estimated at the PB is less than10 mW/mm2 (www.stanford.edu/group/dlab/cgi-bin/graph/chart.php), and a similar range has been used by most researchers and by us earlier19,55,62,63. Note that this is probably a high estimate because some light is probably lost at the interface between the fiber-optic cable and the implanted optic-fiber.
Data analysis
Latency and respiratory data analysis: EEG arousals in response to CO2 were identified by EEG transition from NREM to a waking state, which was usually accompanied by EMG activation, as described previously. The latency of all the EEG arousals after onset of stimulation were scored and were compared across the Laser-ON and Laser-OFF days. Respiratory data was analyzed by running the respiratory script in the Spike2 (CED, UK) software, which performs breath by breath analysis for many respiratory parameters such as RR, VT and MV. For both latency and respiratory data, the ranges for analysis were selected by individuals that were blind to the treatment groups, who based the selection off the following criteria: 1. Trials with at least 30 seconds of NREM sleep before CO2/ stimulation; 2. Select 5 breaths before CO2 or laser stimulation, during CO2/ stimulation before arousal and then at post- arousal for respiratory analysis; 3. Exclude trials with REM sleep.
Fiber photometry data: The voltage GCaMP and UV signals were Gaussian low-pass filtered at 4 Hz, and saved for offline analysis. Both signal channels (465 and 405 nm) were monitored continuously throughout recordings, with the 405 nm signal (UV) used as an isobestic control. Signals detected with 405 nm wavelength light are not calcium-dependent and are indicative of background fluorescence or motion artifacts. A change in fluorescence (ΔF/F) was calculated by normalizing F to baseline fluorescence. For generating heat maps, min-max normalization was performed that causes linear transformation and the data is scaled in the range (0,1).
Inscopix calcium-image processing: Calcium recording files were spatially filtered and motion corrected to correct the rigid brain movements using the Inscopix data processing (IDPS ver1.6). To extract the calcium activity traces from the individual cells, we used manually drawn small regions of interest. Raw traces were converted to ΔF/F (F-F baseline average/ F baseline average), where F was the fluorescent at any given point and F baseline average was the average baseline fluorescence. These baseline image calculations were performed by the IDPS to derive ΔF/F value for each cell.
Statistical analysis: All statistical analyses were performed using SigmaPlot 12.3 (Systat Software, Inc.). For statistical comparisons, we first confirmed if the data meets with the assumptions of the ANOVA, then either one way or two-way ANOVA was performed to compare the effects between various treatment groups. If differences in the mean values among the treatment groups were greater than would be expected by chance; then all pairwise multiple comparisons were performed using the Holm-Sidak method. The F and P values are described in the results section with details of the statistical tests also given in the respective figure legends and represented in the figures. The ‘n’ is reported in the figures and results and represents the number of animals, and the error bars represent mean± SEM. Using SigmaPlot 12.3, we also tested the sample size and power of the tests post hoc and found that the power of each statistical test was at least 80% at alpha= 0.05, suggesting adequate sample sizes for all the experiments. A probability of error of less than 0.05 was considered significant.